It is well established that excessive oxidative stress due to free radicals may injure biological tissues. The natural defenses of cells and tissues revolve around antioxidant mechanisms that have evolved to protect the cells and tissues against high levels of oxidative stress. In our oxygen rich atmosphere the presence of oxygen at certain times of stress may be injurious; this has been termed the oxygen paradox and relates to the role of oxygen in generating and participating in free radical processes. In certain disease states associated with periods of restricted blood flow to tissues, such as heart attack, stroke and restricted flow to the extremities, intermittent episodes of no flow followed by re-flow of blood constitute ischemia/reperfusion (I/R) oxidative stress.
In my laboratory I have utilized cardiac cells such as endothelial cells and cardiomyocytes and their respective membranes as in vitro models to test the susceptibility to free radical stress. I have utilized one of the peptides (glutathione) that is rich in sulfhydryl moieties as an indicator of oxidative stress particularly in the cultured endothelial cell model. In the presence of high levels of superoxide and other radicals glutathione is consumed as a defense against injury to vital membranes. Glutathione is in the cytosol and to some extent in the membrane. I have used Vitamin E or .alpha.-tocopherol and other antioxidant agents to provide "first line defense" against excessive radical injury. I have shown that in the presence of these exogenous antioxidant agents the endogenous protective mechanism of glutathione preservation is maintained against levels of oxidative stress that would exhaust the glutathione levels if higher "pharmacological levels" of antioxidants were not present. Thus, this in vitro assay system has enabled us to determine whether an agent has potent antioxidant properties.
Excessive Free Radical Production in Mg-deficiency:
In my studies of dietary deficiency of magnesium in animals we have established that excessive production of free radicals occurs. In these animals one of the first indicators of depletion of normal endogenous levels of antioxidants is seen using the red cell glutathione assay. After just a few weeks on a diet deficient in magnesium we have found significant decreases in red cell glutathione levels. When these animals are supplemented with antioxidants (such as .alpha.-tocopherol and antioxidant drugs such as probucol), I have been able to observe protection of a red cell glutathione levels. These studies showed the absence of magnesium resulted in such high levels of free radical production. What seems to be the causal mechanism is that neurological peptides are triggered to be released by low circulating blood levels of magnesium and these in turn trigger production of free radicals by white blood cells, endothelial cells, macrophages and other cells that are capable of responding to neuropeptide stimulation by producing radicals. Further, I have discovered that nitric oxide is produced in excess in these Mg deficient animals; this is another form of a free radical.
Clinical Trials with Magnesium Therapy and Relevant Animal Studies:
In a large investigation, the Second Leicester Intravenous Magnesium Intervention Trial, Woods and colleagues found that giving 2 to 3 grams of magnesium sulfate over first five minutes of presentation and then another 5 grams over the next 24 hours reduced mortality by myocardial infarction at 28 days by 24% and lowered the incidence of left ventricular failure by 25% as reported in Lancet vol. 343, page 1553, 1992.
The Fourth International Study of Infarct Survival (ISIS-4) clinical trials also raised controversy in cardiology with regard to the efficacy of intravenous magnesium administration in patients with acute myocardial infarction.
With the emerging data from the Limit 2 clinical trial and animal studies showing protection of ischemic myocardium, the role of pharmacological levels of magnesium (both clinically as well as in the animal laboratory) came under active investigation. Some of the data showed that mortality was improved in patients who received intravenous magnesium for chest pain (indicating heart attack) in the emergency room. The conclusion of Limit 2 trial was that magnesium given early to patients with infarction was beneficial. The clinical trial by Schecter, et al. Amer. Heart J. vol. 132, No. 2, part 2 483-486 (1996) also confirmed the efficacy of magnesium at pharmacological levels when given intravenously to patients having heart attacks. Using animal models Herzog, et al. Lancet vol. 343, pages 1285-1286 (1994) provided convincing evidence that magnesium, when present during reperfusion (minute to hour) of previously ischemic myocardial tissue, was protective; indeed, a decrease in the size of the anticipated myocardial infarction was reported in these animal studies. These essential observation of these animal studies (which were more tightly controlled in their design than previous clinical trials such as Limit 2 and ISIS 4) pointed to the protective effect of pharmacological levels of magnesium during the early stage of reperfusion injury. In my previous studies with the isolated perfused rat heart and in the in vivo pig heart, as well as in coronary bypass patients, I have shown a burst of oxygen derived free radicals and free radical derived products in the effluent from hearts perfused after periods of I/R. The earliest burst occurs within seconds to minutes of reperfusion and is not observed beyond 30 minutes of the reperfusion period. The hypothesis that emerged from these observations is that oxygen derived free radicals participate in the I/R injury and that if magnesium at pharmacological levels is able to protect the myocardium during reperfusion it may have an antiradical effect.
Intravenous Therapy with Magnesium:
Clinically Mg sulfate has been utilized for a number of years for patients with toxemia of pregnancy. The intravenous use of Mg sulfate in these patients is efficacious in lowering hypertension which is life threatening in some of these patients. Recent data suggest that the health of the fetus also benefits from magnesium therapy in the peripartum period of time. Another clinical use of intravenous Mg sulfate is in the coronary care unit where patients who have life threatening arrhythmias particularly Torsade de Pointe are given intravenous infusions of Mg sulfate to block these arrhythmias; some of these patients may be deficient in magnesium and repletion is effective in controlling the disordered heart beat. It is curious that only intravenous Mg sulfate is utilized in this country. Other countries have preparations of Mg chloride which can be given intravenously. Early studies by Selye, et al. Amer. Heart J. 55: 163-173 (1958) suggested that Mg sulfate might not be as effective as Mg chloride or other magnesium preparations with different anions. No data exist on Mg gluconate in such clinical studies.
Potential Mechanisms for Cytoprotection by Mg gluconate and Proposed Clinical Efficacy:
The present invention relates to the fact that Mg gluconate has therapeutic efficacy greater than that of Mg sulfate in pathobiological conditions that result from excessive free radical production in vivo. In particular, I/R injury of the myocardium and cerebral tissues is one area of efficacy. Another area is that of cardioplegia at the time of bypass surgery. I/R is also observed in organ preservation; the harvesting of cardiac, renal and hepatic tissues is associated with a prolonged period of no flow or anoxia; prior to implantation in the recipient reinstitution of flow occurs with an oxygenated solution that may result in free radical induced membrane injury. The present invention relates to the use of intravenous Mg gluconate in the early phases of myocardial infarction, bypass cardioplegia, stroke, organ preservation for transplantation and other acute I/R injury conditions. Mg gluconate has greater efficacy than the use of Mg sulfate.